Modeling and Molecular Dynamics of Membrane Proteins

Transcription

1 Modeling and Molecular Dynamics of Membrane Proteins Emad Tajkhorshid Department of Biochemistry, Center for Biophysics and Computational Biology, and Beckman Institute University of Illinois at Urbana-Champagin

2 Why Do Living Cells Need Membrane Channels (Proteins)? Living cells also need to exchange materials and information with the outside world however, in a highly selective manner. Extracellular (outside) Cytoplasm (inside)

3 Phospholipid Bilayers Are Excellent Materials For Cell Membranes Hydrophobic interaction is the driving force Self-assembly in water Tendency to close on themselves Self-sealing (a hole is unfavorable) Extensive: up to millimeters

4 Lipid Diffusion in a Membrane D lip = 10-8 cm 2.s -1 (50 Å in ~ 5 x 10-6 s) D wat = 2.5 x 10-5 cm 2.s -1 Modeling mixed lipid bilayers! Once in several hours! (~ 50 Å in ~ 10 4 s) ~9 orders of magnitude slower ensuring bilayer asymmetry

5 Fluid Mosaic Model of Membrane Lateral Diffusion Allowed Flip-flop Forbidden Ensuring the conservation of membrane asymmetric structure

6 Technical difficulties in Simulations of Biological Membranes Time scale Heterogeneity of biological membranes 60 x 60 Å Pure POPE 5 ns ~100,000 atoms

7 Coarse-grained modeling of lipids 150 particles 9 particles! Also, increasing the time step by orders of magnitude.

8 by: J. Siewert-Jan Marrink and Alan E. Mark, University of Groningen, The Netherlands

9 Protein/Lipid ratio Pure lipid: insulation (neuronal cells) Other membranes: on average 50% Energy transduction membranes (75%) Membranes of mitocondria and chloroplast Purple membrane of halobacteria Different functions = different protein composition

10 Protein / Lipid Composition The purple membrane of halobacteria

11 Gramicidin A Might be very sensitive to the lipid head group electrostatic and membrane potential

12 Central cavity

13 Analysis of Molecular Dynamics Simulations of Biomolecules A very complicated arrangement of hundreds of groups interacting with each other Where to start to look at? What to analyze? How much can we learn from simulations? It is very important to get acquainted with your system

14 Aquaporins Membrane water channels

15 Monomeric pores Water, glycerol, Tetrameric pore Perhaps ions???

16 Functionally Important Features Tetrameric architecture Amphipatic channel interior Water and glycerol transport Protons, and other ions are excluded Conserved asparagine-prolinealanine residues; NPA motif Characteristic half-membrane spanning structure N ~100% conserved -NPA- signature sequence E NPA E NPAR C

17 A Semi-hydrophobic channel

18 Molecular Dynamics Simulations Protein: ~ 15,000 atoms Lipids (POPE): ~ 40,000 atoms Water: ~ 51,000 atoms Total: ~ 106,000 atoms NAMD, CHARMM27, PME NpT ensemble at 310 K 1ns equilibration, 4ns production 10 days /ns 32-proc Linux cluster 3.5 days/ns O2000 CPUs 0.35 days/ns 512 LeMieux CPUs

19 Protein Embedding in Membrane Hydrophobic surface of the protein Ring of Tyr and Trp

20 Embedding GlpF in Membrane 77 A

21 112 A 122 A

22 A Recipe for Membrane Protein Simulations Align the protein along the z-axis (membrane normal): OPM, Orient. Decide on the lipid type and generate a large enough patch (MEMBRANE plugin in VMD, other sources). Size, area/lipid, shrinking. Overlay the protein with a hydrated lipid bilayer. Adjust the depth/ height to maximize hydrophobic overlap and matching of aromatic side chains (Trp/Tyr) with the interfacial region Remove lipids/water that overlap with the protein. Better to keep as many lipids as you can, so try to remove clashes if they are not too many by playing with the lipids. Add more water and ions to the two sides of the membrane (SOLVATE / AUTOIONIZE in VMD) Constrain (not FIX) the protein (we are still modeling, let s preserve the crystal structure; fix the lipid head groups and water/ion and minimize/simulate the lipid tails using a short simulation.

23 A Recipe for Membrane Protein Simulations Continue to constrain the protein (heavy atoms), but release everything else; minimize/simulate using a short constantpressure MD (NPT) to pack lipids and water against the protein and fill the gaps introduced after removal of protein-overlapping lipids. Watch water molecules; They normally stay out of the hydrophobic cleft. If necessary apply constraints to prevent them from penetrating into the open cleft between the lipids and the protein. Monitor the volume of your simulation box until the steep phase of the volume change is complete (.xst and.xsc files). Do not run the system for too long during this phase (over-shrinking; sometimes difficult to judge). Now release the protein, minimize the whole system, and start another short NPT simulation of the whole system. Switch to an NP n AT or an NVT simulation, when the system reaches a stable volume. Using the new CHARMM force field, you can stay with NPT.

24 Lipid-Protein Packing During the Initial NpT Simulation

25 Adjustment of Membrane Thickness to the Protein Hydrophobic Surface

26 Glycerol-Saturated GlpF

27

28 Description of full conduction pathway

29 Complete description of the conduction pathway Constriction region Selectivity filter

30 Channel Hydrogen Bonding Sites {set frame 0}{frame < 100}{incr frame}{ } animate goto $frame set donor [atomselect top name O N and within 2 of (resname GCL and name HO) ] lappend [$donor get index] list1 set acceptor [atomselect top resname GCL and name O and within 2 of (protein and name HN HO) ] lappend [$acceptor get index] list2

31 Channel Hydrogen Bonding Sites GLN 41 OE1 NE2 TRP 48 O NE1 GLY 64 O ALA 65 O HIS 66 O ND1 LEU 67 O ASN 68 ND2 ASP 130 OD1 GLY 133 O SER 136 O TYR 138 O PRO 139 O N ASN 140 OD1 ND2 HIS 142 ND1 THR 167 OG1 GLY 195 O PRO 196 O LEU 197 O THR 198 O GLY 199 O PHE 200 O ALA 201 O ASN 203 ND2 LYS 33 HZ1 HZ3 GLN 41 HE21 TRP 48 HE1 HIS 66 HD1 ASN 68 HD22 TYR 138 HN ASN 140 HN HD21 HD22 HIS 142 HD1 GLY 199 HN ASN 203 HN HD21HD22 ARG 206 HE HH21HH22

32 Channel Hydrogen Bonding Sites GLN 41 OE1 NE2 TRP 48 O NE1 GLY 64 O ALA 65 O HIS 66 O ND1 LEU 67 O ASN 68 ND2 ASP 130 OD1 GLY 133 O SER 136 O TYR 138 O PRO 139 O N ASN 140 OD1 ND2 HIS 142 ND1 THR 167 OG1 GLY 195 O PRO 196 O LEU 197 O THR 198 O GLY 199 O PHE 200 O ALA 201 O ASN 203 ND2 LYS 33 HZ1 HZ3 GLN 41 HE21 TRP 48 HE1 HIS 66 HD1 ASN 68 HD22 TYR 138 HN ASN 140 HN HD21 HD22 HIS 142 HD1 GLY 199 HN ASN 203 HN HD21HD22 ARG 206 HE HH21HH22

33 The Substrate Pathway is formed by C=O groups

34 The Substrate Pathway is formed by C=O groups Non-helical motifs are stabilized by two glutamate residues. N E NPA E NPAR C

35 Conservation of Glutamate Residue in Human Aquaporins

36 Glycerol water competition for hydrogen bonding sites

37 Revealing the Functional Role of Reentrant Loops Potassium channel

38 Both from E. coli AqpZ is a pure water channel GlpF is a glycerol channel AqpZ vs. GlpF We have high resolution structures for both channels

39 Steered Molecular Dynamics is a non-equilibrium method by nature A wide variety of events that are inaccessible to conventional molecular dynamics simulations can be probed. The system will be driven, however, away from equilibrium, resulting in problems in describing the energy landscape associated with the event of interest. Second law of thermodynamics W ΔG

40 Jarzynski s Equality Transition between two equilibrium states λ = λ i T λ = λ(t) T work W heat Q λ = λ f e -βw p(w) ΔG W W ΔG Δ G = G G f i p(w) C. Jarzynski, Phys. Rev. Lett., 78, 2690 (1997) C. Jarzynski, Phys. Rev. E, 56, 5018 (1997) e βw = e βδg In principle, it is possible to obtain free energy surfaces from repeated nonequilibrium experiments. β = 1 k T B

41 Steered Molecular Dynamics constant force (250 pn) constant velocity (30 Å/ns)

42 SMD Simulation of Glycerol Passage Trajectory of glycerol pulled by constant force

43 Constructing the Potential of Mean Force 4 trajectories v = 0.03, Å/ps k = 150 pn/å f ( t) = k[ z( t) z0 vt] W ( t) dtʹ vf( tʹ ) = t 0

44 Features of the Potential of Mean Force Captures major features of the channel The largest barrier 7.3 kcal/mol; exp.: 9.6±1.5 kcal/mol Jensen et al., PNAS, 99: , 2002.

45 Periplasm Cytoplasm Features of the Potential of Mean Force Asymmetric Profile in the Vestibules Jensen et al., PNAS, 99: , 2002.

46 Artificial induction of glycerol conduction through AqpZ Y. Wang, K. Schulten, and E. Tajkhorshid Structure 13, 1107 (2005)

47 Three fold higher barriers periplasm cytoplasm SF NPA AqpZ 22.8 kcal/mol GlpF 7.3 kcal/mol Y. Wang, K. Schulten, and E. Tajkhorshid Structure 13, 1107 (2005)

48 Could it be simply the size? Y. Wang, K. Schulten, and E. Tajkhorshid Structure 13, 1107 (2005)

49 It is probably just the size that matters! Y. Wang, K. Schulten, and E. Tajkhorshid Structure 13, 1107 (2005)

50 Water permeation 5 nanosecond Simulation 18 water conducted In 4 monomers in 4 ns water/monomer/ns Exp. = ~ 1-2 /ns 7-8 water molecules in each channel

51

52 Correlated Motion of Water in the Channel Water pair correlation The single file of water molecules is maintained.

53 Diffusion of Water in the channel One dimensional diffusion: 2 Dt = ( z z ) Experimental value for AQP1: e-5 t 0 2

54 Diffusion of Water in the channel 2 Dt = ( z z ) t Time (ns) Improvement of statistics

55 Water Bipolar Configuration in Aquaporins

56 Water Bipolar Configuration in Aquaporins

57

58 R E M E M B E R: One of the most useful advantages of simulations over experiments is that you can modify the system as you wish: You can do modifications that are not even possible at all in reality! This is a powerful technique to test hypotheses developed during your simulations. Use it!

59 Electrostatic Stabilization of Water Bipolar Arrangement

60 Proton transfer through water H O H O H O H + H + H + H + H + H H H H + H + O H O H O H H + H + H H H H + H O H O H O H + H + H + H H H H +

61 Cl - channel K + channel Aquaporins

62

63 A Complex Electrostatic Interaction SF NPA Surprising and clearly not a hydrophobic channel M. Jensen, E. Tajkhorshid, K. Schulten, Biophys. J. 85, 2884 (2003)

64 A Repulsive Electrostatic Force at the Center of the Channel QM/MM MD of the behavior of an excessive proton

65 Combining all-atom and coarsegrained models to simulate transport across lipid bilayers

66 Peptide aggregation and Pore formation in lipid bilayers

67 Alamethicin 20-residue peptide No charge forming pores in the membrane 20 residue antimicrobial peptide

68 CG molecular systems allow for time scales of 3-4 orders of magnitude longer, because: Significant reduction of the degrees of freedom (or number of interacting particles/beads) Softer potentials allowing much longer time steps µm length scale and µs time scale Bilayer, micelle, and vesicle formation Fusion of bilayers and vesicles,

69 Alamethicin 20-residue peptide No charge forming pores in the membrane 20 residue antimicrobial peptide

70 Simulation Setting 49 peptides in 288 DMPC All-atom model equilibrated 1ns Converted to a CG model Simulated for 1µs 0.5 µs snapshot was reversecorarse-grained to an all atom model All atom model simulated for 20 ns

71 Coarse-Graining Four-to-one mapping P N C Q Employs CHARMM-like force fields parameterized using all-atom simulations

72 L. Thogersen, et al., Biophysical Journal (2008) Peptide Aggregation

73 g (R ) for peptides A g (R ) init t =1_s R / Å Number of clusters B Time / _s Av. number of neighbors C Time / _s MSD / Å D lipid peptide Time / _s

74 What brings the helices together?

75 L. Thogersen, et al., Biophysical Journal (2008) Peptide Insertion

76 Strong perturbation of the bilayer structure BUT no water permeation/pore formation!?

77 Hydration of the head group region in coarse-grained and all-atom models

78 Reverse Coarse-Graining X X

79 Reverse Coarse-Graining Mapping back CG beads to all-atom clusters Re-solvating the system 5000 steps of minimization Simulated annealing for 20 ps (T changing from 610K to 300K, ΔT = -10K) while constraining atoms to the position of the corresponding CG beads

80 Hydration of the head group region in coarse-grained and all-atom models

81 Alemethicin-induced Membrane Poration

82 Alemethicin-induced Membrane Poration

83 Alemethicin-induced Membrane Poration